QC Report


general
Report generated at2025-02-25 00:22:59
TitlePancreas
DescriptionATAC-seq data from Human ENCODE pancreas data processed in 2025
Pipeline versionv2.2.0
Pipeline typeatac
Genomehg38
Alignerbowtie2
Sequencing endedness{'rep1': {'paired_end': True}, 'rep2': {'paired_end': True}}
Peak callermacs2

Alignment quality metrics


SAMstat (raw unfiltered BAM)

rep1rep2
Total Reads138334834425693838
Total Reads (QC-failed)00
Duplicate Reads00
Duplicate Reads (QC-failed)00
Mapped Reads135923011413925919
Mapped Reads (QC-failed)00
% Mapped Reads98.397.2
Paired Reads138334834425693838
Paired Reads (QC-failed)00
Read169167417212846919
Read1 (QC-failed)00
Read269167417212846919
Read2 (QC-failed)00
Properly Paired Reads133009924406375972
Properly Paired Reads (QC-failed)00
% Properly Paired Reads96.295.5
With itself134206540409714240
With itself (QC-failed)00
Singletons17164714211679
Singletons (QC-failed)00
% Singleton1.21.0
Diff. Chroms128713233817
Diff. Chroms (QC-failed)00

Marking duplicates (filtered BAM)

rep1rep2
Unpaired Reads00
Paired Reads59783571180149877
Unmapped Reads00
Unpaired Duplicate Reads00
Paired Duplicate Reads926119325493700
Paired Optical Duplicate Reads6742692702838
% Duplicate Reads15.491214.1514

Filtered with samtools flag 1804 (samtools view -F 1804):


Fraction of mitochondrial reads (unfiltered BAM)

rep1rep2
Rn = Number of Non-mitochondrial Reads124125942397055770
Rm = Number of Mitochondrial Reads1236205717962177
Rm/(Rn+Rm) = Frac. of mitochondrial reads0.090572483226162610.0432804825185066

SAMstat (filtered/deduped BAM)

rep1rep2
Total Reads98571138307055396
Total Reads (QC-failed)00
Duplicate Reads00
Duplicate Reads (QC-failed)00
Mapped Reads98571138307055396
Mapped Reads (QC-failed)00
% Mapped Reads100.0100.0
Paired Reads98571138307055396
Paired Reads (QC-failed)00
Read149285569153527698
Read1 (QC-failed)00
Read249285569153527698
Read2 (QC-failed)00
Properly Paired Reads98571138307055396
Properly Paired Reads (QC-failed)00
% Properly Paired Reads100.0100.0
With itself98571138307055396
With itself (QC-failed)00
Singletons00
Singletons (QC-failed)00
% Singleton0.00.0
Diff. Chroms00
Diff. Chroms (QC-failed)00

Filtered and duplicates are removed. Subsampling with atac.subsample_reads is not done in alignment steps. Nodup BAM is converted into a BED type (TAGALIGN) later and then TAGALIGN is subsampled with such parameter in the peak-calling step.

Fragment length statistics (filtered/deduped BAM)

rep1rep2
Fraction of reads in NFR0.31451501987554720.35047792539723627
Fraction of reads in NFR (QC pass)FalseFalse
Fraction of reads in NFR (QC reason)out of range [0.4, inf]out of range [0.4, inf]
NFR / mono-nuc reads1.2096154094257591.5135908362866812
NFR / mono-nuc reads (QC pass)FalseFalse
NFR / mono-nuc reads (QC reason)out of range [2.5, inf]out of range [2.5, inf]
Presence of NFR peakTrueTrue
Presence of Mono-Nuc peakTrueTrue
Presence of Di-Nuc peakTrueTrue

rep1
rep1
rep2
rep2

Open chromatin assays show distinct fragment length enrichments, as the cut sites are only in open chromatin and not in nucleosomes. As such, peaks representing different n-nucleosomal (ex mono-nucleosomal, di-nucleosomal) fragment lengths will arise. Good libraries will show these peaks in a fragment length distribution and will show specific peak ratios.



Sequence quality metrics (filtered/deduped BAM)

rep1
rep1
rep2
rep2

Open chromatin assays are known to have significant GC bias. Please take this into consideration as necessary.


Annotated genomic region enrichment

rep1rep2
Fraction of Reads in universal DHS regions0.59739196680472530.4024372657499235
Fraction of Reads in blacklist regions0.00073605724223250830.0012917115451050402
Fraction of Reads in promoter regions0.34447674734160010.1737267825119087
Fraction of Reads in enhancer regions0.315614323129758350.32585977743247346

Signal to noise can be assessed by considering whether reads are falling into known open regions (such as DHS regions) or not. A high fraction of reads should fall into the universal (across cell type) DHS set. A small fraction should fall into the blacklist regions. A high set (though not all) should fall into the promoter regions. A high set (though not all) should fall into the enhancer regions. The promoter regions should not take up all reads, as it is known that there is a bias for promoters in open chromatin assays.


Library complexity quality metrics


Library complexity (filtered non-mito BAM)

rep1rep2
Total Fragments55558177174037843
Distinct Fragments49493558154341567
Positions with Two Read462439814686568
NRF = Distinct/Total0.8908420.886828
PBC1 = OneRead/Distinct0.8934380.890795
PBC2 = OneRead/TwoRead9.5621989.36139

Mitochondrial reads are filtered out by default. The non-redundant fraction (NRF) is the fraction of non-redundant mapped reads in a dataset; it is the ratio between the number of positions in the genome that uniquely mapped reads map to and the total number of uniquely mappable reads. The NRF should be > 0.8. The PBC1 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with AT LEAST one read pair. PBC1 is the primary measure, and the PBC1 should be close to 1. Provisionally 0-0.5 is severe bottlenecking, 0.5-0.8 is moderate bottlenecking, 0.8-0.9 is mild bottlenecking, and 0.9-1.0 is no bottlenecking. The PBC2 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with EXACTLY two read pairs. The PBC2 should be significantly greater than 1.


Fragment: read for a single-ended dataset, pair of reads for a paired-ended dataset
NRF: non redundant fraction
PBC1: PCR Bottleneck coefficient 1
PBC2: PCR Bottleneck coefficient 2
PBC1 is the primary measure. Provisionally


Replication quality metrics


IDR (Irreproducible Discovery Rate) plots

rep1_vs_rep2
rep1_vs_rep2
rep1-pr1_vs_rep1-pr2
rep1-pr1_vs_rep1-pr2
rep2-pr1_vs_rep2-pr2
rep2-pr1_vs_rep2-pr2
pooled-pr1_vs_pooled-pr2
pooled-pr1_vs_pooled-pr2

Reproducibility QC and peak detection statistics

overlapidr
Nt217013139090
N1205373148487
N2257215169303
Np266980184782
N optimal266980184782
N conservative217013139090
Optimal Setpooled-pr1_vs_pooled-pr2pooled-pr1_vs_pooled-pr2
Conservative Setrep1_vs_rep2rep1_vs_rep2
Rescue Ratio1.23024887909940881.3285067222661586
Self Consistency Ratio1.25242850812911131.1401873564689164
Reproducibility Testpasspass

Reproducibility QC


Number of raw peaks

rep1rep2
Number of peaks242579299607

The number of peaks is capped at 300000
Peaks are called from macs2 with p-val threshold 0.01

Peak calling statistics


Peak region size

rep1rep2idr_optoverlap_opt
Min size150.0150.0150.0150.0
25 percentile311.0292.0629.0412.0
50 percentile (median)641.0560.01005.0751.0
75 percentile1095.01044.01402.01232.0
Max size4361.03711.03680.03680.0
Mean753.274665160628720.60068689983881050.0259711443755865.589961794891

rep1
rep1
rep2
rep2
idr_opt
idr_opt
overlap_opt
overlap_opt

Enrichment / Signal-to-noise ratio


TSS enrichment (filtered/deduped BAM)

rep1rep2
TSS enrichment27.2398213643703815.94762653291641

rep1
rep1
rep2
rep2

Open chromatin assays should show enrichment in open chromatin sites, such as TSS's. An average TSS enrichment in human (hg19) is above 6. A strong TSS enrichment is above 10. For other references please see https://www.encodeproject.org/atac-seq/


Jensen-Shannon distance (filtered/deduped BAM)

rep1rep2
AUC0.123167068914079080.24041904351035
Synthetic AUC0.496305810309423450.49790422743066864
X-intercept0.120160408457827810.09267466709847408
Synthetic X-intercept0.00.0
Elbow Point0.87670498957464970.7768695161957767
Synthetic Elbow Point0.50025465150108720.5021212278421999
Synthetic JS Distance0.58760738846199210.38480852769397506

Peak enrichment


Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

rep1rep2rep1-pr1rep2-pr1rep1-pr2rep2-pr2pooledpooled-pr1pooled-pr2
Fraction of Reads in Peaks0.5342800140949980.31616044617564710.52327646814270380.30907369561419460.52108603070172590.308893024631946230.37480687838828610.367014060441055560.3652654210499228

FRiP for overlap peaks

rep1_vs_rep2rep1-pr1_vs_rep1-pr2rep2-pr1_vs_rep2-pr2pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks0.344350485710582240.5198421164621230.30375886310755470.36550431634238206

FRiP for IDR peaks

rep1_vs_rep2rep1-pr1_vs_rep1-pr2rep2-pr1_vs_rep2-pr2pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks0.29930058520284080.485388309101189460.26531924552141720.3311399939137118

For macs2 raw peaks:


For overlap/IDR peaks: